Courtney McCorstin TA Alex Pilote October 1 2014 Enzymes Strange Brew How Effective is Amylase Objective to determine how quickly amalyse can break down starch An important step in beer production is the breakdown of starch from grain into sugars that are used for fermentation This process is called malting and companies generally use enzymes to help speed up this process A local brewery found a cheaper source of amylase a major enzyme in this production This would help decrease the cost of production but before using this the brewery would have to figure out if amylase did break down the starch and how efficiently it worked My general approach was to create a blank using Tris and Lugol s reagent To make a standard curve of concentration vs absorbance a 1 2 dilution series will be made using a starch solution and Tris This standard curve will determine how long it would take for a sample of amylase enzyme to metabolize starch into sugar The absorbency of these dilutions will be tested with the spectrophotometer A mixture of amylase and starch will be made and timed in order to stop reaction rates at certain intervals up to 5 minutes Each test tube of starch and amylase will be tested for its absorbance and concentration determined A concentration vs time graph will then be made to find out the rate of reaction for how amylase breaks down starch First we created a blank solution negative control in a test tube using 4mL of Tris and 1mL of Lugol s Reagent We were then able to set the absorbance of the spectrophotometer using this blank as a calibration There were three big test tubes so in the first one we put 15mL of starch and 15mL of Tris Since we were given a 0 2g L concentration and wanted a 0 1g L concentration we then diluted 2 fold with Tris This came out to be 4mL of this solution with 0mL of Tris 2mL of this solution with 2mL of Tris and 1mL of this solution with 3mL of Tris In the second big test tube experimental we put 14 7mL of Tris 0 3mL of Amylase which we only added right before we were about to start the experiment and 15mL of starch We added 1mL of Lugol s to 5 test tubes and then added 4mL of solution in big test tube 2 Since Lugol s stopped the reaction we had to set a timer so we knew when exactly to add it At 0 minutes we added 4mL of this solution into one of the 5 test tubes with Lugol s in it At 1 minute we added 4mL of this solution to Lugol s and continued with this pattern until the 5th test tubes reaction was finished After the 5 test tubes were filled we got their absorbance from the spectrophotometer The third big test tube was used to make sure that amylase was actually breaking down the starch We put 15mL of starch 0 3mL of amylase and 14 7mL of Tris into the third big test tube Then repeated what we did for the second big test tube however no amylase was added We placed 4mL of test tube 3 solution into a test tube of 1mL of Lugol s at 0 minutes and then 4mL again at 1 minute and continued on until the 5 test tubes were filled Courtney McCorstin TA Alex Pilote October 1 2014 Results Big Test Tube 1 Dilutions We used the formula M1V1 M2V2 to find the final concentration of starch in the starch tris solution 0 2g L 15mL x 30mL x 0 1M Dilutions in 5 test tubes Test tube 1 0 1g L 4mL x 4mL Test tube 2 0 1g L 2mL x 4mL Test tube 3 0 1g L 1mL x 4mL Test tube 4 0 1g L 0 5mL x 4mL Test tube 5 0 1g L 0 25mL x 4mL x 0 1M x 0 05M x 0 025M x 0 0125M x 0 00625M Big Test Tube 2 We used the formula M1V1 M2V2 to figure out how much of each substance to put in this test tube We were told to use a final concentration of 0 1mg mL for both amylase and starch How much amylase to use 10mg mL x 0 1g L 30mL x 0 3mL of amylase How much starch to use 0 2g L x 0 1g L 30mL x 15mL The final volume is 30mL so 30 15 0 3 x amount of Tris which comes out to be 14 7mL of Tris Absorbance Values Starch Tris dilutions from the 5 test tubes For Standard Curve Concentrations g L Absorbance nm Blank 0 0 0 1 1 29 0 05 0 578 0 025 0 258 0 0125 0 109 0 00625 0 033 Courtney McCorstin TA Alex Pilote October 1 2014 Test Tube 2 starch tris amylase solution At every minute we added 1mL of Lugol s Time minutes Absorbance nm 0 1 2 3 4 1 16 1 005 0 840 0 712 0 662 Concentration from standard curve 0 1041 0 091 0 076 0 063 0 058 Test Tube 3 starch tris NO amylase this one contains only Tris and starch with 1mL of Lugol s being added every minute Time minute Absorbance nm Concentration from standard curve 0 1 36 0 1221 1 1 31 0 119 2 1 33 0 121 3 1 31 0 119 4 1 32 0 120 We were able to find the concentrations of the solutions with the amylase and without the amylase by using the recorded absorbance values We looked at our standard curve and traced from where the absorbances were located until it fell upon our best fit line This showed us what the concentrations were The point of this lab was to find the rate at which the enzyme amylase broke down starch The concentrations for the starch tris amylase solution were 0 1041 at 0 minutes 0 091 at 1 minute 0 076 at 2 minutes 0 063 at 3 minutes and 0 058 at 4 minutes These results demonstrate that amylase is breaking down the starch This is further reinforced by the fact that there is no change in the concentration of the starch tris solution that contained no amylase The concentration of this solution at 0 minutes was 0 1221 while at 4 minutes it was 0 120 This is very little change compared to the starch tris solution that contained amylase We did this by using the formula later concentration earlier concentration time interval Which comes out to be 0 058g L 0 1041g L 4 minutes 0 012g L min Based on the results that we got it shows that the concentration of the starch decreases over time in the presence of amylase We concluded that amylase does break down starch and would function as a good enzyme to metabolize starch into sugar in the brewing of beer Before the company switches to amylase however they should compare these results to what enzyme they …